CD19⁻ BMPC express IgG with moderately mutated V_H gene rearrangements and secrete antibodies commonly found in blood serum. (A) BMPC were analyzed for expression of CD19 and cyt IgG, IgM, and IgA. Summarized results from 11 donors are shown in (B) and were compared using the Mann-Whitney U test, ***P < .001. Median values are indicated. (C-D) Individual fluorescence-activated cell sorter (FACS)-sorted CD19⁺ and CD19⁻ BMPC from 3 individuals were subjected to nested single-cell reverse transcriptase-polymerase chain reaction for amplification of V_HDJ_HC gene rearrangements.¹⁸  A total of 82 sequences from CD19⁺ PC and 72 sequences from CD19⁻ BMPC were analyzed: 33 CD19⁺IgG⁺, 44 CD19⁻IgG⁺, 46 CD19⁺IgA⁺, and 24 CD19⁻IgA⁺ sequences (and 7 IgM sequences, not separately shown). Sequences were analyzed for immunoglobulin isotype (supplemental Figure 2B) for V_H and J_H family use. Distributions were compared by χ² test; respective P values are indicated (C), for absolute numbers and frequencies of somatic mutations per V_H sequence, and for CDR3 length and mutations located within RGYW/WRCY hotspot motifs (D). Data from 65 tetanus toxoid (TT)-specific PB¹⁸  from blood 1 week after intramuscular TetDiph vaccination from 3 donors, respectively, are shown for comparison. After χ² tests did not show significant differences in V_HJ_HC segment use and CDR3 length distribution among different individuals’ samples, data were pooled and displayed together. Mann-Whitney U test was used to analyze data in (D) (*P < .05; **P < .01; ***P < .001). Median values are indicated. (E) Frequencies of antibody-secreting PC specific for TT were determined by Elispot among 4 pairs of FACS-sorted CD19⁺ and CD19⁻ BMPC (donors were aged 37, 52, 62, and 72 years). Median values are indicated.